This proposal investigates the questions of the specificity and regulation of RNA splicing using bioinformatics, genomics and RNA interference. Identification of sequence elements that are important for RNA splicing, such as intronic and exonic enhancers/ silencers, is critical for gene recognition and interpretation of genomic analysis of human disease genes. Further, regulation of alternative splicing of genes such as CD44 controls cell growth and motility processes important for development and cancer. Synthesis of splicing isoforms of CD44 is stimulated by Ras activation and in a positive freedback loop, these isoforms, when bound to HGF, activate Ras. The RESCUE-ESE program was used, to identify a set of hexamer sequences with properties of exonic enhancers of RNA splicing. We have further shown that single nucleotide polymorphism (SNPs) in the human genome which alter these ESEs are under selective pressure. We propose to extend these studies and use gene silencing by RNAi to identify proteins important for the activities of these ESEs. Initially, the degree of inclusion of exons containing an ESE will be quantitated by microarray analysis of a pool of reporter vectors after transfection of cells silenced by RNAi for a specific gene. RNAi methods, gene silencing by a lentivirus vector constructed to express a hairpin RNA that is processed to an siRNA (Lentihair) or direct application of siRNAs, will be used to silence a set of candidate proteins. The SRprotein family will be the first set of candidate genes examined. These experiments will reveal subgroups of ESEs which depend functionally on a common protein for activity. Subsequently, libraries of Lentihair recombinants will be used for silencing of other genes important for the functionally defined subgroups of ESEs. Regulation of alternative RNA splicing will be directly studied by RNAi gene silencing. These CD44 isoforms have different functions when expressed on the surface of cells. The constitutive form of the protein is thought to suppress cell growth and motility while some of the isoforms containing variable exons are associated with cell growth and motility. We have recently found that the inclusion of this set of CD44 exons is dependent upon the SRml60 protein, a coactivator of RNA splicing and Sam68, an Src substrate. We propose to investigate this alternative splicing system using Lentihair gene silencing.